To deal effectively with cases where environmental pollution results from various pollutants, it is necessary to know how pollutants have been degraded or how the degradation has proceeded. In cases where the environment has been polluted with biodegradable pollutant sources such as crude oil, a method of analyzing the concentration of various hydrocarbons in crude oil has conventionally been used to grasp the progress of the biological degradation of crude oil etc. as pollutant sources.
However, the method of analyzing various hydrocarbons in crude oil was problematic in that accurate analysis results are hardly obtained in spite of cumbersome and time-consuming procedures.
Further, such analysis of hydrocarbons in crude oil requires complex instruments, so there are few cases where analytical instruments are installed at a site of environmental sanitation. Accordingly, analytical samples after collection should be stored and transported in many cases to a site where they can be analyzed. However, because these samples are deteriorated during storage and transportation, there are also cases where the obtained results are not reproducible or fail to reflect the state of biological degradation of crude oil at the polluted site.
Hydrocarbons contained in crude oil etc. are divided roughly into saturated hydrocarbons, aromatic hydrocarbons, resin, and asphaltene. Among these components, any of the aromatic hydrocarbons, resin, and asphaltene contain aromatic compounds and thus hardly undergo degradation by microorganisms. Accordingly, the concentration of these aromatic components in crude oil can be used as an indication of the progress of degradation of pollutants such as crude oil etc. However, it is difficult to grasp which components in pollutants such as crude oil etc. have been degraded and which components are not degraded and remain.
Because analysis of aromatic hydrocarbon compounds in crude oil is difficult as described above, it was proposed to use methods of identifying microorganisms degrading hydrocarbons in crude oil in order to know the state of biological degradation in crude oil. In the conventional methods of identifying microorganisms by biological examination based on e.g. assimilability of saccharides, however, there is the problem that accurate results cannot necessarily be obtained in spite of many examination items and cumbersome and time-consuming procedures.
For highly accurate detection of microorganisms having specific functions, a method of utilizing the recent analysis at the gene level using a nucleotide sequence in 16S rRNA from a microbial species is also conceivable. However, because 16S rRNA is a gene possessed by every microorganism, it is difficult to detect only a group of microorganisms having specific metabolic mechanism. For detection using such a gene as 16S rRNA common to microorganisms, it is necessary to conduct the complicated procedures in which a specific nucleotide sequence in 16S rRNA is amplified by PCR and each of the amplified fragments is then cloned and sequenced. In addition, there is less difference in the nucleotide sequence of 16S rRNA among microorganisms of the same genus or species, so it is necessary to determine its long nucleotide sequence by use of a plurality of primers.